L-ascorbic acid has been produced from 2-keto-L-gulonic acid by the well-known Reichstein method (Helv. Chim. Acta 17, 311-328 (1934)). The method has been used commercially for more than 60 years with many chemical and technical modifications to improve the efficiency of each of the steps to the compounds D-glucose, D-sorbitol, L-sorbose, diacetone-L-sorbose, diacetone-2-keto-L-gulonic acid, 2-keto-L-gulonic acid, and methyl 2-keto-L-gulonate, and L-ascorbic acid. In this process, the conversion of D-sorbitol to L-sorbose is the sole microbial step, the others being chemical steps. The conversion of diacetone-2-keto-L-gulonic acid into L-ascorbic acid is achieved by two different procedures: 1) deprotection to give 2-keto-L-gulonic acid, followed by esterification with methanol and base-catalyzed cyclization; and 2) acid-catalyzed cyclization to L-ascorbic acid directly from the protected or deprotected 2-keto-L-gulonic acid. The starting material for base-catalyzed reactions is methyl 2-keto-L-gulonate, itself prepared by treatment of the acid with acidic methanol. An alternative reaction of the methyl ester with sodium bicarbonate or sodium acetate produces sodium L-ascorbate. Many chemical and technical modifications have improved the efficiency of each step, enabling the multistep synthesis to remain the principally used and economical process.
D-Erythorbic acid has been produced from D-glucose via 2-keto-D-gluconic acid, which itself can be produced by fermentation with a strain belonging to the genus Pseudomonas, and methyl 2-keto-D-gluconate. D-Erythorbic acid is mainly used as an antioxidant for food additives.
Much time and effort has been devoted to finding other methods of synthesizing L-ascorbic acid by microorganisms. Most microbial productions of L-ascorbic acid have been focused on the production of an intermediate of L-ascorbic acid production, 2-keto-L-gulonic acid, from L-sorbose (G. Z. Yin et al., Wei Sheng Wu Hsueh Pao. 20, 246-251 (1980); A. Fujiwara et al., EP 213 591 (Roche); T. Hoshino et al., U.S. Pat. No. 4,960,695 (Roche); and I. H. Nogami et al., EP 221 707), from D-sorbitol (A. Fujiwara et al., EP 213 591 (Roche); T. Hoshino et al., U.S. Pat. No. 5,312,741 (Roche); M. Niwa et al., WO 95/23220; and S. F. Stoddard et al., WO 98/17819), or from D-glucose via 2,5-diketogluconic acid with a single, mixed or recombinant culture (T. Sonoyama et al., Appl. Environ. Microbiol. 43, 1064-1069 (1982); and S. Anderson et al., Science 230, 144-149 (1985)). The 2-keto-L-gulonic acid can then be converted into L-ascorbic acid by chemical means as described above.
The involvement of an enzymatic process for the conversion of the 2-keto-L-gulonic acid ester into L-ascorbic acid has recently been reported (J. C. Hubbs, WO 97/43433 (Eastman Chemical Company)). WO 97/43433 describes a process for allegedly preparing L-ascorbic acid by contacting 2-keto-L-gulonic acid or an ester thereof with a hydrolase enzyme catalyst selected from the group consisting of a protease, an esterase, a lipase and an amidase. Using a hydrolase such as a protease, an esterase, a lipase or an amidase, WO 97/43433 exemplifies the formation of L-ascorbic acid from an ester of 2-keto-L-gulonic acid (butyl 2-keto-L-gulonate), but no apparent formation of L-ascorbic acid from 2-keto-L-gulonic acid itself. WO 97/43433 discloses, for example, that Candida antartica B lipase catalyzed the reaction to form 413-530 mg/l of methyl 2-keto-L-gulonate, but no L-ascorbic acid, from 1% 2-keto-L-gulonic acid in the presence of 8.6% methanol at pH 3.1-3.2 at 38.degree. C. Ester synthetic activity of Candida antartica B lipase on 2-keto-L-gulonic acid, an .alpha.-keto-carboxylic acid, at acidic pH is apparently positive, but intramolecular ester formation by this lipase was negligible. It does not disclose a lactonase as the hydrolase enzyme catalyst for the purpose of producing L-ascorbic acid from 2-keto-L-gulonic acid.
Surprisingly, it has now been found that the conversion of 2-keto-L-gulonic acid to L-ascorbic acid can be performed by a lactonase enzyme. Accordingly, it has been surprising found that the selectivity of lactonase on cyclic esters is favorable for the production of L-ascorbic acid from 2-keto-L-gulonic acid.
Many kinds of lactonases are known, including gluconolactonase (EC 3.1.1.17) of Escherichia coli (F. Hucho et al., Biochem. Biophys. Acta 276, 176-179 (1972)) or of Zymomonas mobilis (M. Zachariou et al., J. Bacteriol. 167, 863-869 (1986) and V. Kanagasundaram et al., Biochem. Biophys. Acta 1171, 198-200 (1992)), and lactonohydrolase of Fusarium oxysporum (S. Shimizu et al., Eur. J. Biochem. 209, 383-390 (1992)). Further reported lactonases include L-arabinonolactonase (EC 3.1.1.15) and D-arabinonolactonase (EC 3.1.1.30) of Pseudomonas saccharophilia, L-rhamnono- 1,4-lactonase (EC 3.1.1.65) of Pullularea pullulans, xylono-1,4-lactonase (EC 3.1.1.68) of Pseudomonas fragi and Gluconobacter oxydans, cellobinolactonase of Trichoderma reesei (Chem.-Ztg. 113, 122-124 (1989)), 1,4-lactonase (EC 3.1.1.25), and lactonases with the EC numbers 3.1.1.19, 3.1.1.24, 3.1.1.27, 3.1.1.36, 3.1.1.37, 3.1.1.38, 3.1.1.39, 3.1.1.45, 3.1.1.46, and 3.1.1.57.
Among the lactonases, the lactonohydrolase of Fusarium oxysporum has been developed for the industrial asymmetric hydrolysis of D-pantoyl lactone (K. Sakamoto et al., U.S. Pat. No. 5,275,949). The enzyme catalyzes the hydrolysis of a relatively broad range of lactone compounds, including D-pantoyl lactone and several aldonolactones, e.g. D-glucono-.delta.-lactone and D-galactono-.gamma.-lactone, and the reverse reaction, lactonization.
Nucleotide sequences are available for the genes of some lactonases, i.e., the gluconolactonase gene of Zymomonas mobilis (960 bp; 320 amino acid residues; V. Kanagasundaram et al., Biochem. Biophys. Acta 1171, 198-200 (1992)) and the lactonohydrolase gene of Fusarium oxysporum (1,140 bp; 380 amino acid residues; K. Sakamoto et al., WO 97/10341).